Rho Kinase as a Target
The Rho family of small GTP binding proteins can be activated by several extracellular stimuli such as growth factors, hormones and mechanic stress and function as a molecular signaling switch by cycling between an inactive GDP-bound form and an active GTP-bound form to elicit cellular responses. Rho kinase (RHO KINASE) functions as a key downstream mediator of Rho and exists as two isoforms (RHO KINASE1 and RHO KINASE2) that are ubiquitously expressed. RHO KINASES are serine/threonine kinases that regulate the function of a number of substrates including cytoskeletal proteins such as adducin, moesin, Na+—H+ exchanger 1 (NHE1), LIM-kinase and vimentin, contractile proteins such as the myosin light chain phosphatase binding subunit (MYPT-1), CPI-17, myosin light chain and calponin, microtubule associated proteins such as Tau and MAP-2, neuronal growth cone associate proteins such as CRMP-2, signaling proteins such as PTEN and transcription factors such as serum response factor (Loirand et al, Circ Res 98:322-334 (2006)). RHO KINASE is also required for cellular transformation induced by RhoA. As a key intermediary of multiple signaling pathways, RHO KINASE regulates a diverse array of cellular phenomena including cytoskeletal rearrangement, actin stress fiber formation, proliferation, chemotaxis, cytokinesis, cytokine and chemokine secretion, endothelial or epithelial cell junction integrity, apoptosis, transcriptional activation and smooth muscle contraction. As a result of these cellular actions, RHO KINASE regulates physiologic processes such as vasoconstriction, tissue remodeling, inflammation, edema, proliferative disorders, neurite extension/retraction, and neurodegeneration.
The use of prototype non-potent Rho-kinase inhibitors, Y27632 or fasudil, in animal models has suggested a number of potential benefits of Rho-kinase inhibitors. Y27632 has shown favorable activity in animal models of respiratory disorders such as airway hyperreactivity and asthma (Schaafsma et al. Respiratory Research 7:121-127, 2006), airway remodeling and idiopathic pulmonary fibrosis (Shimizu et al. Am J Respir Crit. Care Med 163:210-217, 2001) and RSV infection (Hashimoto et al. Thorax 57:524-527, 2002). Fasudil has been shown to have favorable activity in models of asthma (Taki F et al. Clin Exp Allergy, 37:599-607, 2007); pulmonary hypertension (Shimokawa et al. Arterioscler Thromb Vasc Biol 25:1767-1775, 2005).
Glaucoma
Glaucoma is an ophthalmic disease that leads to irreversible visual impairment. It is the fourth most common cause of blindness and the second most common cause of vision loss in the United States, and the most common cause of irreversible vision loss among African-Americans. Generally speaking, the disease is characterized by a progressive optic neuropathy caused at least in part by deleterious effects resulting from increased intraocular pressure. In normal individuals, intraocular pressures range from 12 to 20 mm Hg, averaging approximately 16 mm Hg. However, in individuals suffering from primary open angle glaucoma, intraocular pressures generally rise above 22 to 30 mm Hg. In angle closure or acute glaucoma intraocular pressure can reach as high as 70 mm Hg leading to blindness within only a few days. Interestingly, the loss of vision can result from statistically normal intraocular pressures in individuals with unusually pressure-sensitive eyes; a condition known as normotensive glaucoma. [See, e.g., P. L. Kaufman and T. W. Mittag, “Medical Therapy Of Glaucoma,” Ch. 9, Sec. II (pp. 9.7-9.30) In P. L. Kaufman and T. W. Mittag (eds.): Glaucoma (Vol. 7 of S. M. Podos and M. Yanoff (eds): Textbook of Ophthalmology Series). London, Mosby-Year Book Europe Ltd. (1994); A. C. Guyton, Textbook of Medical Physiology (W. B. Saunders Co., Sixth Ed.), pp. 386-89 (1981)].
Open-angle glaucoma constitutes approximately 90% of all primary glaucomas and is characterized by abnormally high resistance to fluid (aqueous humor) drainage from the eye. Normal resistance is required to maintain an intraocular pressure sufficient to maintain the shape of the eye for optical integrity. This resistance is provided by the trabecular meshwork, a complex, multilaminar tissue consisting of specialized cells with a dense actomyosin cytoskeletal network, collagenous beams and extracellular matrix. The resistance of the trabecular meshwork normally is such that intraocular pressure is ˜16 mm Hg, a pressure at which aqueous humor leaves the eye at the same rate at which it is produced (2.5 μL/minute). In the glaucomatous eye, the rate of aqueous humor production remains constant, while it is the increased resistance to outflow that is responsible for the elevated intraocular pressure.
Typical treatments for glaucoma comprise a variety of pharmaceutical approaches for reducing intraocular pressure (TOP), each with their drawbacks. Beta-blockers and carbonic anhydrase inhibitors reduce aqueous humor production, which is needed to nourish the avascular lens and corneal endothelial cells, and the prostaglandins affect the uvealscleral outflow pathway, which only accounts for 10% of the total outflow facility. There are currently no commercially approved therapeutic agents which act directly upon the trabecular meshwork, the site of aqueous humor drainage where increased resistance to aqueous humor outflow is responsible for elevated IOP. Therefore, a medical need remains for improved IOP-lowering medications that target this structure. Pharmacological agents which target the trabecular meshwork may provide relief to the significant numbers of patients that do not respond adequately to current IOP-lowering medications and/or cannot tolerate the side effects associated with these agents. Additionally, these molecules may prove beneficial as adjunctive therapy in combination with other classes of IOP-lowering medications.
U.S. Pat. Nos. 6,586,425, 6,110,912, and 5,798,380 disclose a method for the treatment of glaucoma using compounds that affect the actin filament integrity of the eye to enhance aqueous humor outflow. These patents also specifically disclose kinase inhibitors as well as latrunculin-A, latrunculin-B, swinholide-A, and jasplakinolide, which cause a perturbation of the actin cytoskeleton and tight junctional complexes in the trabecular meshwork or the modulation of its interactions with the underlying membrane. Perturbation of the cytoskeleton and the associated adhesions reduces the resistance of aqueous humor flow through the trabecular meshwork and thereby reduces intraocular pressure.
Wound healing is another approach in which these classes of molecules can aid in modulating IOP. Trabeculectomy is the most common form of glaucoma filtration surgery and remains as the first-line therapy for surgical reduction of pharmacologically uncontrolled intraocular pressure in primary open angle glaucoma. This procedure establishes a limbal fistula through which aqueous humor drains into the subconjunctival space establishing a filtering bleb to lower intraocular pressure. The success of the procedure is highly dependent on pharmacological modulation/inhibition of wound healing.
A major advance in the surgical management of glaucoma has been the use of antimetabolites to prevent scarring after glaucoma filtration surgery. Postoperative scarring of the filtering bleb is the most crucial factor in determining the short and long-term outcome of modern glaucoma filtration surgery. The antimetabolites mitomycin C (MMC) and 5-fluorouracil (5-FU) are widely used to suppress scarring and thus failure of the filtering bleb. In a large retrospective study, conventionally performed trabeculectomy has shown a failure rate of up to 30% within 3 months after surgery. To lower the incidence of this detrimental complication, various methods have been investigated in order to avoid scarring of the filtering bleb, mostly dealing with the intraoperative or postoperative application of antimetabolic drugs
Despite their positive long-term effect on prolonged filtration, the application of cytotoxic drugs to a surgically opened eye increases the incidence of severe complications such as concomitant increases in vision threatening complications. MMC exhibits a high incidence of severe post-application complications, as does 5-FU; although its side effects mainly affect the corneal epithelium its clinical use is limited by severe pain and discomfort to the patient. No sufficient method has been established to achieve satisfying postoperative long-term surgical results with only minimal or no side effects for the patient.
Allergic Conjunctivitis
Allergic eye disease primarily affects the conjunctiva. The signs and symptoms include itching, tearing, conjunctival edema, hyperemia, watery discharge, burning, and photophobia. Symptoms are usually bilateral; however, one eye can be affected more than the other. The most common allergic eye disease, allergic conjunctivitis (AC) can be subdivided into acute, seasonal and perennial. All three types result from classic Type I IgE-mediated hypersensitivity (Abelson, M B., et. al. Surv Ophthalmol; 38(S):115, 1993).
Two phases of the ocular allergic response have been indentified. The immediate response to allergens is mediated predominantly by mast cells, which are present in high concentrations in the normal conjunctiva, and increase further in patients with AC (Tsubota, K, et al., Cornea, 10:525, 1991). Mast cells become activated when allergen-IgE cross linking occurs, and chemical mediators are released by exocytosis. Histamine, the main mediator of the early response, causes vasodilatation, vasopermeability, and itching. Mast cells also release a variety of cytokines and chemokines, resulting in the influx of other inflammatory cells and continued inflammation, representing the late phase of the allergic reaction. Eosinophils, basophils, and neutrophils appear 6 to 10 hours after allergen challenge, followed by lymphocytes and monocytes.
Allergic conjunctivitis is a relatively benign ocular disease of young adults (average age of onset of 20 years of age) that causes significant suffering and use of healthcare resources, although it does not threaten vision. Ocular allergy is estimated to affect 20 percent of the population on an annual basis, and the incidence is increasing (Abelson, M B et. al., Sury Ophthalmol., 38(S):115, 1993). AC impacts productivity and while there are a variety of agents available for the treatment of AC, numerous patients still lack good control of symptoms and some are tolerating undesired side effects. Surveys have shown 20% of patients with AC are not fully satisfied with their AC medications and almost 50% feel they receive insufficient attention from their physicians (Mahr, et al., Allergy Asthma Proc, 28(4):404-9, 2007).
Corneal Hyposensitivity and Neurodegeneration
An undesirable effect following laser photorefractive keratectomy (PRK), laser-assisted-in-situ keratomileusis (LASIK), and keratoplasty, is a functional reduction of corneal sensitivity, which occurs from approximately 3 weeks to one year and is due to severing of the corneal nerves during surgery. For example, it has been reported that the corneal nerve is apparently severed after LASIK (Tuuli U, et al., Experimental Eye Research 66: 755-763, 1998), and the corneal sensitivity decreases in a corneal region where, after LASIK, neurogram is not observed or the nerve bundle is too short to create connection (Tuuli U, et al., Investigative Ophthalmology & Visual Sciences, 41: 393-397, 2000). It has been demonstrated that the corneal hyposensitivity after PRK and LASIK causes lower lacrimal gland response and decreased lacrimal fluid (Ang R T, et al., Current Opinion in Ophthalmology 12: 318-322, 2001). As a result of the hypofunction of corneal sensitivity, patients after a corneal surgery blink less number of times, problematically showing the symptoms of dry eye. Additionally, in the patients with dry eye, lacrimal hypofunction gives rise to corneal hyposensitivity, which, upon combination with further lacrimal hypofunction, problematically aggravates the sensory component of the corneal surface. At present, recovery of corneal hyposensitivity following corneal surgery is left to spontaneous recovery, and in the treatment of dry eye, no active treatment is provided to recover corneal sensitivity. Moreover, while corneal hyposensitivity is caused by the diseases accompanying corneal neurodegeneration, such as neuroparalytic keratopathy, corneal ulcer, diabetic keratopathy and the like, no appropriate treatment is available at present.
Corneal hyposensitivity is caused by the diseases accompanying corneal neurodegeneration, such as neuroparalytic keratopathy, corneal ulcer, diabetic keratopathy and the like. Rho is a low molecular weight G protein included in the Rho family (containing Rho, Rac, Cdc42, etc.), and is known to be involved in actin cytoskeleton organization and neurite retraction reaction. C3 enzyme, a Rho protein inhibitor, is known to extend cell protrusion of 3T3 fibroblast (Hirose, M. et al., The Journal of Cell Biology, 141: 1625-1636, 1998), and a method of promoting the growth of central nerve axon by the administration of an effective amount of Rho protein inhibitor to patients is disclosed (JP-T-2001-515018 and EP-1,011,330-A). In addition, a Rho kinase inhibitor, which is among the effector molecules of Rho protein, is known to have an axon extension action of retinal ganglion cells, and exhibit a regeneration promoting action on the optic nerve cell (WO 02/83175 and EP-1,142,585-A). WO 03/020281 teaches that a compound capable of promoting nerve regeneration or neurite extension can be used for the treatment of a disease state caused by a corneal nerve disorder after surgery such as LASIK and the like. As to the trigeminal nerve, it has been reported that, in a rat trigeminal nerve tissue culture (trigeminal tract in whole mount cultures) system, extension of neurotrophin-induced nerve axon of nerve growth factor (NGF) and the like is inhibited by a Rho activator (lysophosphatidic acid), and facilitated by introduction of dominant negative Rho into a cell (Ozdinler, P. Hande et al., The Journal of Comparative Neurology, 438:377-387, 2001).
Dry Eye
There are many ocular conditions where it is therapeutically desirable to correct improper tear fluid production. Dry eye is the general term for disease abnormalities that impact the pre-corneal tear film leading to a loss of mucous-containing goblet cells of the conjunctiva and eventually desquamation of the corneal epithelium that leads to destabilization of the cornea-tear interface (Gilbard J et al. CLAO Journal 22(2), 141-45 (1996)). There are several main structures responsible for maintaining the properties of the tear film such as the glands and ducts surrounding the eye and the ocular surface. These structures maintain the tear film via regulation of water and electrolyte transport and via mucin release by goblet cells. Among the ocular conditions where disruption of one of these structures can cause or lead to “dry eye disease” are: keratoconjunctivitis sicca (KCS), age-related dry eye, Stevens-Johnson syndrome, Sjogren's syndrome, ocular cicatrical pemphigoid, blepharitis, corneal injury, infection, Riley-Day syndrome, congenital alacrima, nutritional disorders or deficiencies, pharmacologic side effects, eye stress and glandular and tissue destruction, environmental exposure to smog, smoke, excessively dry air, airborne particulates, autoimmune and other immunodeficient disorders, and comatose patients rendered unable to blink. This is not to be considered an exhaustive list but is used to describe some of the diseases that can lead to dry eye disease.
Treatment for dry eye disease is effective regulation of the tear film. This can be accomplished by enhancing natural production or improving flow from the glands surrounding the eye or applying artificial tears to the ocular surface. The glands can be blocked due to inflammation of the surrounding tissue or the duct and gland itself. Blockage due to inflammation can be seen by increases in pro-inflammatory cytokines, redness and puffiness on and surrounding the ocular surface. Reduction of this inflammation can help return tear production to normal function and improve corneal health. (Wilson S et al. American Academy of Ophthalmology 114(1), 76-79 (2007)).
Currently, the pharmaceutical treatment of dry eye disease is mostly limited to administration of artificial tears (saline solution) to temporarily rehydrate the eyes and to reduction of inflammation ((Riento K et al. Nat Rev Mol Cell Biol, 4:446-456, 2003)). However, artificial tears, the most widely used group of products, often have contraindications and incompatibility with soft contact lenses (Lemp M et al. Cornea 9(1), S48-550 (1990)).
Macular Edema and Degeneration
Macular edema is a condition that occurs when damaged (or newly formed) blood vessels leak fluid onto the macula, a critical part of the retina for visual acuity, causing it to swell and blur vision. Macular edema is a common problem in diabetic retinopathy, where retinal vessel injury causes edema. Edema also occurs in the proliferative phase of diabetic retinopathy, when newly formed vessels leak fluid into either, or both, the macula and/or vitreous. Macular edema is commonly problematic in age-related macular degeneration (wet form) as well, where newly formed capillaries (angiogenesis) leak fluid into the macula.
Age related macular degeneration (AMD) is a progressive eye condition affecting as many as 10 million Americans. AMD is the number one cause of vision loss and legal blindness in adults over 60 in the U.S. As the population ages, and the “baby boomers” advance into their 60's and 70's, a virtual epidemic of AMD will be prevalent. The disease affects the macula of the eye, where the sharpest central vision occurs. Although it rarely results in complete blindness, it robs the individual of all but the outermost, peripheral vision, leaving only dim images or black holes at the center of vision.
Macular degeneration is categorized as either dry (atrophic) or wet (neovascular). The dry form is more common than the wet, with about 90% of AMD patients diagnosed with dry AMD. The wet form of the disease usually leads to more serious vision loss. In the dry form, there is a breakdown or thinning of the retinal pigment epithelial cells (RPE) in the macula, hence the term “atrophy”. These RPE cells are important to the function of the retina, as they metabolically support the overlying photoreceptors.
The clinical hallmark of atrophic AMD is accumulation of macular drusen, yellowish deposits just deep to the retinal pigment epithelium (“RPE”). Histopathologic examination of eyes with atrophic AMD reveals deposition of lipid and proteinaceous material deep to the RPE in Bruch's membrane. Drusen formation occurs naturally with age, with ocular exposure to visible light and UV light, metabolic changes of ocular cells related to age, and the formation of lipofuscin. Genetic predisposition can also factor into drusen formation. The formation of drusen can result in local inflammation as extracellular debris forms around the RPE, photoreceptors, and other ocular structures. The immune response which results brings about a number of components, one of which is membrane attack complex. The membrane attack complex can cause the death of host cells, which would include the RPE and photoreceptors. As a consequence, more cellular debris and drusen form as a result of the local inflammatory response, perpetuating the cycle (Nowak J Z Pharmacol Rep. 58(3):353-363, 2006).
In aged eyes with AMD, Bruch's membrane is often about 3 times thicker than normal. This thickening is thought to be comprised of lipid as well as modified and cross-linked protein, which impedes transport of nutrients across Bruch's membrane from the choriocapillaries to the outer retina. This thickened barrier comprised of lipid and cross-linked protein impedes transport of nutrients across Bruch's membrane from the choriocapillaries to the outer retina. At present, there is no proven effective treatment for dry AMD other than the use of multivitamins and micronutrients.
Wet AMD occurs when new vessels form and grow through Bruch's membrane into the sub-RPE and subretinal space. This neovascular tissue is very fragile and hyperpermeable. Frequently, it bleeds causing damage to the overlying retina. As the blood organizes, functional macular tissue is replaced by scar tissue. To prevent vision loss, it would be desirable to intervene therapeutically prior to the development of neovascularization.
AMD is a challenging disease for both patient and doctor, because there are very few treatment options and, with the exception of anti-oxidants, no proven preventative therapy. While some individuals experience only minor inconvenience from macular degeneration, many others with more severe forms of macular degeneration are incapacitated. Patients may experience a loss of central vision accompanied by metamorphopsia, central scotomas, increased glare sensitivity, decreased contrast sensitivity, and decreased color vision (Rosenburg et al. American Family Physician, 77(10): 1431-1436, 2008). Current therapies, including laser photocoagulation, photodynamic therapy, and anti-angiogenic therapeutics have had mixed results, and, in certain instances, have caused deleterious side effects. A need exists for additional treatments that reduce the effects of macular degeneration and edema.
Proliferative Vitreal Retinopathy
One of the most common causes of retinal detachment is proliferative vitreoretinopathy (PVR), an intraocular, non-malignant cellular proliferation. This process results ultimately in a separation of the retina from the retinal pigment epithelium, or RPE, because of tractional forces applied directly to the inner and outer retinal surfaces. This is the major cause for failure of retinal re-attachment surgery. (Ryan et al. Am J Ophthalmol, 100:188-193, 1985). PVR is characterized by the formation of contractile cellular epiretinal membranes (ERMs) on both sides of the retina. (Clarkson, et al. Am. J, Ophthalmol., 84:1-17, 1977). While the pathobiology of PVR is not clear, it appears that RPE cells are key to the development of these ERM. (Laqua, et al. Am. J. Ophthalmol., 80:602-618, 1975). A large body of evidence supports the concept that previously quiescent RPE cells, when displaced into the vitreous cavity and exposed to the appropriate combination of cytokines, will divide and differentiate. This differentiation results in cells having myofibroblastic characteristics including adhesiveness and contractility. As these membranes form tight adhesions with the retinal surfaces, tractional forces are generated and detachment ensues. (Hiscott, et al. Br. J. Ophthalmol., 68:708-715, 1984). Most evidence indicates retinal tears as the pathway through which RPE cells move in order to enter the vitreous cavity (Hiscott, et al. Br. J. Ophthalmol., 68:708-715, 1984), and there is a clear association between the size of a retinal tear and the incidence of PVR. (Ryan et al. Am. J. Ophthalmol., 100:188-193, 1985). Viable retinal pigment epithelial cells, displaced into the vitreous cavity, are exposed to a wide variety of proteins, cytokines, and chemoattractants. Extracellular matrix proteins have profound effects on cell morphology and behavior (Glaser, et al. Ophthalmology, 100:466-470, 1993). RPE cells, when exposed in vitro to the extracellular matrix proteins and collagens found in the vitreous, change from their typical epithelial cell morphology to a mesenchymal or fibroblast-like morphology (Hay, et al. Cell Biology of Extracellular Matrix, New York, Plenum Press, 1982). The pathobiology of PVR, while not understood completely, involves the exposure of previously quiescent cells to factors which promote abnormal differentiation and cell division. This differentiation results in adhesive cells which contract in an unregulated, disorganized fashion and produce the tractional forces which detach the retina. (Mandelcorn, et al. Am J Ophthalmol, 80:227-237, 1975).
The small GTPase, Rho, regulates the organization of the actin cytoskeleton by promoting the assembly of focal adhesions and actin stress fibers. A family of Rho-associated serine/threonine kinase isozymes named p160RHO KINASE and ROKα/Rho-kinase/RHO KINASE 2 has been identified as a class of Rho effectors that can induce focal adhesions and stress fibers in cultured fibroblasts and epithelial cells in vitro. (Amano M, Chihara K, Kimura K, et al. Science, 275:1308-1311, 1997). In patients with PVR, ERMs are characterized by the diffuse presence of α-smooth muscle actin (α-SMA)-positive myofibroblasts, which is presumed to be dedifferentiated RPE cells. (Casaroli-Marano R P et al. Invest Ophthalmol Vis Sci, 40:2062-2072, 1999). Dense bundles of α-SMA microfilaments forming stress fibers within the myofibroblast were observed by electron microscopy in the ERM of patients with PVR, which strongly suggests that α-SMA substantially contributes to PVR development. (Casaroli-Marano R P, et al. Invest Ophthalmol Vis Sci., 40:2062-2072, 1999). A previous study has shown that the Rho kinase inhibitor Y-27632 suppresses type I collagen gel contraction in RPE cells, probably by suppressing expression of α-SMA, which led to attenuation of PVR in an animal model. (Zheng Y. et al. Invest Ophthalmol Vis Sci., 45(2):668-74, 2004).
The current treatment for PVR is vitreoretinal surgery. Although such treatment often is successful, recurrent vitreoretinal traction may result in redetachment. The resulting retinal detachment sometimes causes permanent impairment of visual function. Pharmacologic and other forms of therapy to inhibit recurrent membrane formation are needed.
Blepharitis
Blepharitis, also known as Lid Margin Disease (LMD), is a non-contagious inflammation of the eyelids that manifests itself through scaling and flaking around the eyelashes, excess sebum production and oily scaly discharge, mucopurulent discharge, and matted, hard crusts around the lashes. Accumulation of crust, discharge or debris on the eyelashes and lid margins creates an ideal environment for overgrowth of the staphylococcal bacteria naturally found on the skin of the eyelids and increases the chance of infection, allergic reaction and tear break down. Blepharitis disturbs the production of the critical, outer lipid layer of the tear film which causes the entire tear to evaporate, resulting in dry eye. A reduced tear quantity doesn't properly dilute bacteria and irritants, nor wash inflammatory products away from the lashes and lid margin, so they accumulate and lead to further inflammation worsening the cycle of disease, with blepharitis, meibomian gland dysfunction and dry eye perpetuating each other.
Routine examination of the eyelids of blepharitis patients shows redness caused by capillary congestion (erythema) as well as crusting of the lashes and lid margins. More detailed inspection using a high magnification slit lamp microscope reveals additional features, including loss of lashes (madarosis), whitening of the lashes (poliosis), scarring and misdirection of lashes (trichiasis), crusting of the lashes and meibomian orifices, eyelid margin ulcers, plugging of the meibomian orifices, and lid irregularity (tylosis).
Blepharitis is a common eye disorder throughout the Unites States and the world. There is an apparently high incidence in the general population based on the frequency of diagnoses in ophthalmologists' offices. It affects people of all ages; however blepharitis caused by seborrhea is seen more often in older patients around the age of fifty. Chronic blepharitis has been associated with occupations in which the hands are dirty for much of the day, since poor hygiene is a risk factor. Acute blepharitis results most commonly from an allergic reaction to a drug or chemical substance. Likewise, exposure to irritants such as chemical fumes, smoke, and environmental pollutants can exacerbate the condition of chronic blepharitis. The use of certain drugs can also cause blepharitis. It has been documented that some patients on cancer chemotherapeutic agents such as 5-fluorouracil develop ocular surface and lacrimal complications, including blepharitis, conjunctivitis, keratitis, and eyelid dermatitis (Eiseman A S et al. Ophthal Plast Reconstr Surg, 19:3:216-224, 2003).
Designing an effective treatment plan for blepharitis can be challenging. Treatment includes good hygiene and relies heavily on the patient as a partner in achieving disease management. Since lid scrubs and hot compresses are required multiple times daily, long-term compliance to produce positive results can be an issue. If left untreated, blepharitis can lead to a more serious condition called ulcerative blepharitis accompanied by eyelid scarring, scarring of the cornea, and eventually loss of visual function.
It is well known that during acute and chronic inflammation various putative mediators of inflammation are released by the inflamed tissues and by leukocytes. The concentrations of these mediators and leukocytes are indicative of the level or degree of inflammation. Likewise, a reduction in concentration of these mediators and leukocytes is an indication of the effectiveness of a drug in treating inflammation. Anti-inflammatory steroidal preparations (e.g., corticosteroids) are currently the drug of choice in the treatment of ocular inflammatory conditions. The use of a topical ophthalmic steroid can be helpful in reducing acute inflammation, however extended use is complicated by severe and numerous side effects. It would be highly desirable to develop new nonsteroidal drugs which have a high therapeutic effectiveness but which do not exhibit steroid-like side effects.
Rho kinase signaling pathways have been implicated in the down regulation of pro-inflammatory pathways (Riento K et al. Nat Rev Mol Cell Biol, 4:446-456, 2003). For example, Rho kinase inhibition by Y-27632 and fasudil in a murine model of airway hyper-reactivity has been shown to reduce the mediators of inflammation (Taki F et al. Clinical and Experimental Allergy, 37:599-607, 2007).
There is a need for an effective or improved method for treating ophthalmic disease such as allergic conjunctivitis, corneal hyposensitivity and kerotopathy, dry eye disease, proliferative vitreal retinopathy, macular edema and degeneration, and blepharitis.
Asthma
Asthma is a common chronic disorder of the airways characterized by variable and recurring symptoms, reversible airway obstruction, bronchial hyperresponsiveness, and an underlying inflammation. Acute symptoms of asthma include cough, wheezing, shortness of breath and nocturnal awakening. These symptoms usually arise from bronchospasm and require and respond to bronchodilator therapy (see Expert Panel Report 3: Guidelines for the Diagnosis and Management of Asthma, NIH Publication No. 07-4051, Bethesda, Md.: U.S. Department of Health and Human Services; National Institutes of Health; National Heart, Lung, and Blood Institute; National Asthma Education and Prevention Program, (2007) and references therein).
Central to the pathophysiology of asthma is the presence of underlying airway inflammation mediated by the recruitment and activation of multiple cell types including mast cells, eosinophils, T lymphocytes, macrophages, dendritic cells and neutrophils. Type 2 T-helper (Th2) cells appear to play a central role in the activation of the immune cascade that results in inflammation. Th2-derived cytokines include IL-5, which is needed for eosinophil differentiation and survival, and IL-4 which is important for Th2 cell differentiation and with IL-13 is important for IgE formation and leads to overproduction of IgE and eosinophilia. IgE-driven activation of mucosal mast cells releases bronchoconstrictor mediators such as histamine and cysteinyl-leukotrienes as well as inflammatory cytokines. Eosinophils contain inflammatory enzymes, generate leukotrienes, and express a wide variety of pro-inflammatory cytokines. Airway epithelial cells also play a role in the inflammatory process via release of cytokines such as eotaxin that direct and modify the inflammatory response. Acute and chronic inflammation can affect not only the airway caliber and airflow but also can increase the existing bronchial hyperresponsiveness to a variety of stimuli, which enhances susceptibility to bronchospasm.
As a consequence of airway inflammation and the generation of growth factors, the airway smooth muscle cell can undergo proliferation, activation, contraction, and hypertrophy—events that can influence airway airflow limitation. In asthma, the dominant physiological event leading to clinical symptoms is airway narrowing and a subsequent interference with airflow. In acute exacerbations of asthma, bronchial smooth muscle contraction (bronchoconstriction) occurs quickly to narrow the airways in response to exposure to a variety of stimuli including allergens or irritants. Allergen-induced acute bronchoconstriction results from an IgE-dependent release of mediators from mast cells that includes histamine, tryptase, leukotrienes, and prostaglandins that directly contract airway smooth muscle. The mechanisms influencing airway hyperresponsiveness are multiple and include inflammation, dysfunctional neuroregulation, and airway remodeling. Airway remodeling involves structural changes including thickening of the sub-basement membrane, subepithelial fibrosis, airway smooth muscle hypertrophy and hyperplasia, blood vessel proliferation and dilation with consequent permanent changes in the airway that increase airflow obstruction and that is not prevented by or fully reversible by current therapies.
Airway epithelium and endothelial cell function are also critically involved in asthma. Upon disease progression, epithelial subbasement membranes thicken with mucus hypersecretion and the formation of inspissated mucus plugs. Changes in endothelial cell integrity lead to edema, another key pathophysiology defining asthmatic change of the airway. These factors serve to further limit airflow and are not directly addressed by current therapies.
Current standard therapies for asthma are a combination of corticosteroids and β2-agonists (anti-inflammatory and bronchodilator drugs). These drugs provide acceptable control of the disease for many asthmatics. However, it is estimated that 5 to 10% of the asthma patients have symptomatic disease despite treatment with this combination of corticosteroids and β2-agonists (Chanez et al., J Allergy Clin Immunol 119:1337-1348 (2007)).
Chronic Obstructive Pulmonary Disease
Chronic obstructive pulmonary disease (COPD) is the most common chronic lung disease associated with significant morbidity and mortality. In the United States, COPD is the fourth leading cause of death and accounts for more than $30 billion in annual health care costs. An estimated 16 million adults are affected by COPD, and each year ˜120,000 Americans die of the disease. COPD is defined as a chronic disease characterized by airway/alveolar/systemic inflammation, with measured airflow obstruction (FEV1/FVC<70% and FEV1<80% predicted) that is partially improved with bronchodilator therapy. The local and systemic release of inflammatory mediators by the lung cells leads to airway disease (chronic obstructive bronchitis) and, in a minority of patients, to destruction of parenchymal tissue (emphysema), both of which can result in the airflow limitation that characterizes COPD (see Doherty D E et al, Clin Cornerstone 6:S5-16 (2004) and MacNee, Clin Ches Med 28:479-513 (2007)). The release of these inflammatory mediators by the lung cells may also exacerbate inflammation in other organ systems, such as that observed in coronary, cerebrovascular, and peripheral vascular conditions.
The chronic inflammation, airway obstruction, and tissue damage that occur in COPD all result from chronic exposure to inhaled toxic substances, primarily cigarette smoke. In response to the chemical insult of inhaled tobacco smoke, inflammatory cells (including macrophages, neutrophils, and T-lymphocytes, primarily CD8 lymphocytes) are activated in the small and large airways as well as in the lung parenchyma. These activated inflammatory cells release a host of cytokines and other mediators (including tumor necrosis factor-α, interleukin [IL]-8, and leukotriene B4), which can cause damage to lung tissue. The end result of the release of these cytokines and mediators may be the development of chronic inflammation of the airways, mucus gland hypertrophy and goblet-cell hyperplasia with increased mucus secretion, fibrosis and narrowing of smaller airways, destruction of the parenchyma (the connective tissue/cells in the lungs), and changes in the blood vessels that may result in the development of pulmonary hypertension. These pathologic changes manifest themselves as mucus hypersecretion, limited airflow, hyperinflation, and gas exchange abnormalities which are the major physiologic abnormalities that characterize COPD. A loss in the integrity of the lung's connective tissue leads to a decrease of elastic recoil and hyperinflation.
Current therapies to treat COPD include bronchodilators, especially anticholinergic agents, that help to some degree decrease hyperinflation, therefore increasing inspiratory capacity and relieving dyspnea. Although corticosteroids are an effective treatment for most cases of asthma, the inflammatory cells and mediators in COPD are not sensitive to treatment with systemic or inhaled corticosteroids making treatment with these agents of limited usefulness in COPD.
RSV Infection
Respiratory syncytial virus (RSV) causes acute respiratory tract illness in persons of all ages. RSV is a leading cause of lower respiratory tract infection (LRTI) in children younger than 2 years. It is associated with up to 120,000 pediatric hospitalizations each year, and is increasing in frequency. RSV also is a significant cause of morbidity and mortality from LRTI in elderly patients (Collins et al., J Virol 82:2040-2055 (2008); Peebles et al., Proc Am Thorac Soc 2:110-115 (2005)).
After replicating in the nasopharynx, RSV infects the small bronchiolar epithelium and extends to the type 1 and 2 alveolar pneumocytes in lung. Pathologic findings of RSV include necrosis of epithelial cells, occasional proliferation of the bronchiolar epithelium, infiltrates of monocytes and T cells centered on bronchial and pulmonary arterioles, and neutrophils between the vascular structures and small airways. This leads to airway obstruction, air trapping and increased airway resistance, and also is associated with a finding of neutrophilia in bronchoalveolar lavage. The immune response to RSV, especially cytokine and chemokine release, appears to play a role in the pathogenesis and severity of bronchiolitis. There is a distinct pattern of cytokines and chemokines induced by RSV infection and some have been associated with disease severity. The cytokines IL-8, IL-6, TNF-alpha, and IL-1 beta can be detected in airway secretions of infected children (Smyth et al. Arch Dis Child 76:210 (1997)), and IL-6 levels correlate with severe disease. Chemokines identified in respiratory tract secretions of children include CCL3, CCL2, CCL11 and CCL5, but only the beta-chemokines, particularly MIP-1 alpha, are associated with severe disease (Welliver et al. Pediatr Infect Dis J 21:457 (2002)).
RSV can involve both lower and upper respiratory tract. Severe lower respiratory tract disease can involve bronchiolitis, bronchospasm, pneumonia, and acute respiratory failure in children. Lower respiratory tract involvement usually occurs with primary infection, and may occur in second infections and can cause wheezing, tachypnea and apnea. Repeat RSV infections occur frequently in children and young adults and result in significant upper respiratory tract symptoms. Signs include cough, coryza, rhinorrhea, and conjunctivitis. RSV infection in adults also may cause short-term airway reactivity.
There is no direct treatment for RSV infection and the respiratory complications it causes. The current therapy for RSV is primarily supportive. Bronchodilator therapy in infants with bronchiolitis, largely caused by RSV infection, did not demonstrate benefit in large randomized trials and systematic reviews. Prophylaxis with palivizumab, a humanized monoclonal antibody, has been indicated for a limited fraction of the pediatric patient population.
Pulmonary Arterial Hypertension
Pulmonary arterial hypertension (PAH) is a disease of the small pulmonary arteries, characterized by vascular narrowing leading to a progressive increase in pulmonary vascular resistance. The consequence of this increased right ventricle after-load is the failure of the afterload-intolerant right ventricle. The pulmonary vascular injury underlying PAH occurs in an idiopathic form or in association with other disease states such as congenital heart disease or COPD. Vasoconstriction, remodeling of the pulmonary vessel wall, and thrombosis contribute to the increased pulmonary vascular resistance in PAH. However, it is now recognized that pulmonary arterial obstruction by vascular proliferation and remodeling is the hallmark of PAH pathogenesis (Humbert et al. J Am Coll Cardiol 43:13 S-24S (2004) and Rubin Proc Am Thorac Soc 3:111-115 (2006)). The process of pulmonary vascular remodeling involves all layers of the vessel wall. Indeed, each cell type (endothelial, smooth muscle, and fibroblast), as well as inflammatory cells and platelets, may play a significant role in PAH. A feature common to all forms of PAH remodeling is the distal extension of smooth muscle into small peripheral, normally nonmuscular, pulmonary arteries within the respiratory acinus. In addition, a hallmark of severe pulmonary hypertension is the formation of a layer of myofibroblasts and extracellular matrix between the endothelium and the internal elastic lamina, termed the neointima.
Pulmonary vasoconstriction is believed to be an early component of the pulmonary hypertensive process. Excessive vasoconstriction has been related to abnormal function or expression of potassium channels and to endothelial dysfunction. Endothelial dysfunction leads to chronically impaired production of vasodilators such as nitric oxide and prostacyclin along with overexpression of vasoconstrictors such as endothelin 1.
Inflammatory mechanisms appear to play a significant role in some types of pulmonary hypertension. Indeed, a subset of PAH patients have circulating autoantibodies including antinuclear antibodies, as well as elevated circulating levels of proinflammatory cytokines IL-1 and IL-6. Lung histology also revealed inflammatory infiltrates (macrophages and lymphocytes) in the range of plexiform lesions in severe PAH as well as an increased expression of chemokines RANTES and fractalkine.
Current therapies for PAH include prostanoids, endothelin receptor antagonists, and phosphodiesterase type V inhibitors. Despite these treatments, the average life expectancy of a PAH patient is generally under five years from the diagnosis of the disease.
Lymphangioleiomyomatosis
Lymphangioleiomyomatosis (LAM) and tuberous sclerosis complex (TSC) are caused by mutations in either of the tuberous sclerosis genes, TSC1 or TSC2, which control cell growth, survival, and motility through the Akt/mammalian target of rapamycin (mTOR) signaling pathway (McCormack Chest 133:507-516 (2008)). Deficiency or dysfunction of the encoded proteins, hamartin or tuberin, respectively, results in a loss of regulation of signals from upstream sources including cell surface tyrosine kinase and G protein coupled receptors. The constitutive activation of mTOR kinase and the downstream S6 kinase (S6K) leads to increased protein translation, and ultimately to inappropriate cellular proliferation, migration, and invasion. These changes lead to smooth muscle cell infiltration and cystic destruction of the lung resulting in progressive dyspnea on exertion, recurrent pneumothoraces, abdominal and thoracic lymphadenopathy, and abdominal tumors, including angiomyolipomas and lymphangiomyomas.
LAM occurs in about 30% of women with tuberous sclerosis complex (TSC) and also in women who do not have TSC (ie, sporadic LAM [S-LAM]). Both S-LAM and TSC-LAM are associated with mutations in tuberous sclerosis genes. In patients with TSC or TSC-LAM, germline mutations in TSC genes are present in all cells of the body and neoplasms and dysplasias occur when somatic TSC mutations result in a loss of heterozygosity for the normal allele. In patients with S-LAM, somatic TSC mutations are confined to lesions in the lung, kidney, and lymph nodes although respiratory involvement predominates.
There are no proven therapies for LAM although bronchodilator therapy is useful for some patients.
Idiopathic Pulmonary Fibrosis
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressives fibrotic disorder of the lower respiratory tract that typically affects adults beyond the age of 40. IPF is thought to occur as a result of initial injury to the lung by environmental factors such as cigarette smoke leading to recruitment of neutrophils, lymphocytes and macrophages to the lung alveoli. Release of fibrogenic cytokines, such as TGF-β□ by alveolar epithelial cells results in fibroblast proliferation, migration, and fibrosis. These fibroblasts not only fill the respiratory space but also secrete collagen and matrix proteins in response to many cytokines leading to parenchymal remodeling (Shimizu et al., Am J Respir Crit Care Med 163:210-217 (2001)). This differentiation of fibroblasts is likely key to the chronic nature of IPF. These events lead to cough and progressive shortness of breath. IPF patients have compromised lung function and have shown restrictive lung volumes and capacities. Although corticosteroids, immunosupressive agents, neutrophil elastase inhibitor, hepatocyte growth factor, and interferon gamma-1b have been proposed as treatment agents for IPF, no treatment other than lung transplantation is known to prolong survival and IPF remains a fatal disorder with a 3 to 6 yr median range of survival (Khalil et al. CMAJ 171:153-160 (2004)). Thus, the first line of treatment of IPF has not yet been established.
Acute Respiratory Distress Syndrome (ARDS) and Ventilator Induced Lung Injury (VILI)
Acute respiratory distress syndrome is a critical illness characterized by acute lung injury leading to permeability pulmonary edema and respiratory failure. ARDS respiratory failure can be caused by various acute pulmonary injuries and is characterized by noncardiogenic pulmonary edema, respiratory distress, and hypoxemia. Despite significant advances in critical care management, overall mortality from ARDS ranges from 25 to 58% (Berstan A D et al. Am J Respir Crit Care Med, 165:443, 2002).
More than 60 causes of ARDS have been identified. A few common causes include sepsis, aspiration of gastric contents, primary bacterial or viral pneumonias, direct chest trauma, ventilator-induced lung injury, prolonged or profound shock, burns, fat embolism, near drowning, massive blood transfusion, transfusion-related lung injury (TRALI), cardiopulmonary bypass, pneumonectomy, acute pancreatitis, inhalation of smoke or other toxic gas, and ingestion of certain drugs (Pepe P et al. Am J Surg, 144:124, 1982; Hudson L D, J Respir Grit Care Med, 151:293, 1995; Zaccardelli D S and Pattishall E N, Grit Care Med, 24:247, 1996; Fowler A et al. Ann Intern Med, 98:593, 1983).
ARDS is described as a “syndrome of acute and persistent inflammation with increased vascular permeability associated with a constellation of clinical, radiological and physiological abnormalities” (Bernard G et al. Am J Respir Grit Care Med, 149:818, 1994; Artigas A et al. Am J Respir Grit Care Med, 157:1332, 1998). The hallmark of ARDS is deterioration in blood oxygenation and respiratory system compliance as a consequence of permeability edema. Whereas a variety of different insults may lead to ARDS, a common pathway probably results in the lung damage and/or failure, leukocyte activation within the lung, along with the release of oxygen free radicals, arachidonic acid metabolites, and inflammatory mediators, resulting in an increase in alveolocapillary membrane permeability. With the loss of this macromolecular barrier, alveoli are flooded with serum proteins, which impair the function of pulmonary surfactant (Said et al. J. Clin. Invest. 44: 458-464; Holm et al. J. Appl. Physio. 63: 1434-1442, 1987). This creates hydrostatic forces that further exacerbate the condition (Jefferies et al., J. Appl. Physio. 64: 5620-5628, 1988), leading to alveolar edema and a concomitant deterioration in gas exchange and lung compliance.
Mechanical ventilation is a common and generally effective means of treating a failing lung. Unfortunately, positive-pressure mechanical support can create or contribute to lung injury (ventilator-induced lung injury, VILI). Mechanical ventilators applying high volumes and pressures can lead to an influx of fluid into the lung. In addition to edema, the injured or ruptured cells trigger a cascade of cellular and biochemical events leading to the inflammation in the lung. Pulmonary sheer stress can develop due to the increased volume as well as due to atelectasis. VILI is also believed to provoke distal airway and alveolar cell inflammation by increasing the production of proinflammatory cytokines. In light of the fact that more than 280,000 Americans are at risk for VILI each year, and mechanical ventilation support and associated intensive care expenditures are estimated in the billions of dollars, VILI is a major public health concern (WO/2007/109582).
Rho kinase signaling pathways are implicated in an array of cellular phenomena many of which play roles in the pathophysiology of ARDS and VILI. These include cytoskeletal rearrangement, actin stress fiber formation, proliferation, chemotaxis, cytokinesis, cytokine and chemokine secretion, endothelial or epithelial cell junction integrity, apoptosis, transcriptional activation and smooth muscle contraction. Several mechanisms such as increased endothelial permeability, inflammatory cell recruitment, and inflammation have been implicated in the pathogenesis of ARDS. Endothelial cells form a major part of the capillary permeability barrier in the lungs and changes are associated with increased capillary permeability (due to endothelial cell contraction and barrier dysfunction; Tinsley J H et al. Am J Physiol Cell Physiol, 279:C1285-1289, 2000). Inflammatory reactions may lead to endothelial paracellular gaps and extravasation of fluid and macromolecules. Airway epithelium can also contribute to inflammation by releasing inflammatory mediators, an event governed in part by Rho signaling (Cummings R J et al. J Biol Chem, 277:30227-30235, 2002).
Cystic Fibrosis (CF)
CF is the most common, life threatening, recessively inherited disease of Caucasian populations, with a carrier rate of 1 in 25 and an incidence of 1 in 2,500 live births. CF is a multisystem disease affecting the digestive system, sweat glands, and the reproductive tract, but progressive lung disease continues to be the major cause of morbidity and mortality (Ratjen, F and Doring, G. Lancet 361:681, 2003). CF patients have abnormal transport of chloride and sodium across the respiratory epithelium, resulting in thickened, viscous airway secretions (Rowe S M et al. N Engl J Med; 352:1992, 2005). Patients develop chronic infection of the respiratory tract with a characteristic array of bacterial flora (Gibson, R L et al. Am J Respir Crit Care Med 168:918, 2003), leading to progressive respiratory insufficiency and eventual respiratory failure. CF is caused by mutations in a single large gene on chromosome 7 that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein (Rommens J M et al. Science; 245:1059, 1989; Collins F S. Science; 256:774, 1992; Drumm, M L et al. Mol Genet Med; 3:33, 1993). CFTR has been shown to function as a regulated chloride channel, which in turn may regulate the activity of other chloride and sodium channels at the cell surface (Boucher R C. Am J Respir Crit Care Med. 150:271-281, 1994). Defective CFTR results in abnormal ion transport and airway surface liquid volume with alterations in the rheology of airway secretions, which become thick and difficult to clear (Wine J J. J Clin Invest; 103:309, 1999). These changes result in reduced mucociliary clearance and a propensity for chronic infection of the respiratory tract with resulting inflammation, progressive airway damage, bronchiectasis, progressive respiratory failure, and death (Mickle J E and Cutting G R. Clinics in Chest Med. 19(3):443-458, 1998).
Respiratory symptoms of CF usually begin early in life (Ratjen, F. and Doring, G. Lancet 361:681, 2003). Respiratory manifestations include recurrent, progressively more persistent cough becoming productive, chronic infection (particularly Pseudomonas aeruginosa), and inflammation leading to progressive tissue damage in the airways. Once infection is established, neutrophils are unable to control the bacteria, even though there is massive infiltration of these inflammatory cells into the lung tissue. Recruited neutrophils subsequently release inflammatory cytokines, reactive oxygen species, and elastase, the latter of which overwhelms the antiproteases of the lung and contributes to progressive destruction of the airway walls. In addition, large amounts of DNA and cytosol matrix proteins are released by degranulating neutrophils, contributing to the increased viscosity of the airway mucus (Davis, P B. Pathophysiology of the lung disease in cystic fibrosis. In: Cystic Fibrosis, Davis, P B (Ed), Marcel Dekker, New York 1993. p. 193). Toxic metabolites released by P. aeruginosa increase the rate of neutrophil apoptosis and decreased removal of apoptotic cells by pulmonary macrophages (Bianchi S M et al. Am J Respir Crit Care Med 177:35-43, 2008), contributing to the accumulation of DNA, protein, and cellular debris in the airway and exacerbating inflammation. Lung damage ultimately advances to the stage of irreversible bronchiectasis (dilated, collapsible airways), leading to progressive air and mucus trapping and ultimate respiratory failure. Other late complications include spontaneous pneumothorax (collapsed lung) and hemoptysis (coughing up blood), which may be massive (Flume P A et al. Chest; 128:720, 2005; Flume P A et al. Chest 128:729, 2005). Terminal findings often include severely congested parenchyma, with grossly purulent secretions in dilated airways. The airway epithelium is hyperplastic, often with areas of erosion and squamous metaplasia. Plugs of mucoid material and inflammatory cells are often present in the airway lumen. Submucosal gland hypertrophy and hyperplasia of airway smooth muscle may also be present (Hays S R et al. Thorax 60:226, 2005.)
Airway hyperreactivity is a common finding in CF patients (Hiatt P et al. Am Rev Respir Dis 137:119, 1988). Many CF patients continue to demonstrate bronchial hyperresponsiveness into adolescence and adulthood, with positive correlations between the degree of airway reactivity and the overall severity of lung disease. The response to bronchodilators does not always persist with increasing age, and some patients demonstrate worsening of expiratory airflow in response to treatment with beta-adrenergic reagents (Gibson, R L et al. Am J Respir Crit Care Med 168:918, 2003). This phenomenon may occur when progressive airway damage leads to a loss of cartilaginous support, resulting in an increased reliance on muscle tone for maintenance of airway patency. Muscle relaxation in this setting can cause collapse of such “floppy” airways, leading to increased airflow obstruction.
The chest radiography may appear normal for an extended period in patients with mild lung disease. As the disease progresses, hyperinflation becomes persistent, and interstitial markings become more prominent. Increasing hyperinflation leads to progressive flattening of the diaphragms, a prominent retrosternal space, and kyphosis (curvature of upper spine) in late stages of disease. Thin-walled cysts may appear to extend to the lung surface, and pneumothorax is observed with increasing frequency in older patients. Computed tomography (CT) of the chest may be helpful in defining the extent of bronchiectasis in some patients (de Jong, P A et al. Radiology 231:434, 2004.]. This is of particular interest in patients who have focal areas of advanced disease, which may sometimes be amenable to surgical resection.
Changes in pulmonary function may be identifiable from a very early age, even before clinical signs of disease are apparent (Long F R et al. J Pediatr 144:154, 2004; Castile R G et al. Pediatr Pulmonol 37:461, 2004). Over time, the majority of CF patients develop an obstructive pattern on pulmonary function testing (PFT). Increases in the ratio of residual volume to total lung capacity (RV/TLC) and decreases in the forced expiratory flow at 25 to 75 percent of lung volume (FEF25-75) provide the most sensitive measures of early airway obstruction. As disease progresses, the forced expiratory volume in one second (FEV1) and the ratio of FEV1 to forced vital capacity (FEV1/FVC) decline (Davis, P B. Pathophysiology of the lung disease in cystic fibrosis. In: Cystic Fibrosis, Davis, P B (Ed), Marcel Dekker, New York 1993. p. 193). The FEV1 is correlated with subsequent survival in CF patients. An FEV1 persistently lower than 30 percent of predicted may be a useful indicator of the need for transplant evaluation in patients who are considered appropriate candidates for that procedure (Kerem E et al. N Engl J Med; 326:1187, 1992). Lung volumes demonstrate increases in total lung capacity (TLC) and residual volume (RV) as hyperinflation progresses. Despite aggressive therapy, baseline pulmonary function gradually decreases as patients get older.
As bronchiectasis and airway obstruction become pronounced, ventilation-perfusion mismatch leads to hypoxemia. This may initially occur only during sleep or exercise, but patients with advanced disease often require continuous oxygen supplementation. Hypercapnia occurs relatively late in the course of CF lung disease. Chronic hypoxemia and hypercapnia may lead to muscular hypertrophy of the pulmonary vasculature, pulmonary hypertension, right ventricular hypertrophy, and eventually cor pulmonale with right heart failure (Eckles M and Anderson P. Semin Respir Crit Care Med 24:323-30, 2003).
Therapeutic intervention for cystic fibrosis includes inhaled and oral antibiotics (tobramycin, azithromycin), bronchodilators (β-adrenergic agonists), DNase I (dornase alpha), hypertonic saline, chest physiotherapy, anti-inflammatory agents (azithromycin, ibuprofen, glucocorticoids), and lung transplantation. Although improved treatment of lung disease has increased survival, the median age for survival is still only 35 years (Cystic Fibrosis Foundation Patient Registry Annual Data Report, 2004), and patients continue to have significant morbidity, including hospitalizations (Ramsey B W. N Engl J Med. 335(3):179-188, 1996).
Bronchiectasis
Bronchiectasis is currently defined as the irreversible and sometimes progressive dilatation and destruction of the bronchial wall caused by a vicious pathogenic cycle of impaired local defense mechanisms, infection, and airway inflammation (Garcia, Arch Bronconeumol, 41(8):407-9, 2005). Bronchiectasis is a syndrome of chronic cough and daily viscid sputum production associated with airway dilatation and bronchial wall thickening. Hemoptysis can also occur. Multiple conditions are associated with the development of bronchiectasis, but all require an infectious insult plus impairment of drainage, airway obstruction, and/or a defect in host defense (Barker, A. F. Clinical manifestations and diagnosis of bronchiectasis. In: UpToDate, King T E (Ed), UpToDate, Wellesley, Mass., 2008).
All types of bronchiectasis are characterized by predominately neutrophilic and mononuclear inflammation with scores of cellular mediators that modulate both acute and chronic inflammatory response and perpetuate the bronchial lesion (Garcia, Arch Bronconeumol, 41(8):407-9, 2005) The ensuing host response, immune effector cells, neutrophilic proteases, reactive oxygen intermediates (eg, hydrogen peroxide [H2O2]), and inflammatory cytokines, causes transmural inflammation, mucosal edema, cratering, ulceration, and neovascularization in the airways. The result is permanent abnormal dilatation and destruction of the major bronchi and bronchiole walls. Recurrent infection is common, which can lead to further scarring, obstruction, and distortion of the airways, as well as temporary or permanent damage to the lung parenchyma (Barker, A. F. Clinical manifestations and diagnosis of bronchiectasis. In: UpToDate, King T E (Ed), UpToDate, Wellesley, Mass., 2008). The characteristic clinical picture is chronic purulent sputum, functional impairment in the form of air flow obstruction, multiple exacerbations of an infectious type that occasionally involve atypical microorganisms, and dyspnea in advanced stages of the disease-all of which cause progressive deterioration of the patient's quality of life (Garcia, Arch Bronconeumol, 41(8):407-9, 2005). Mortality is difficult to estimate given the difficulty in identifying prevalence and the lack of definitive studies. One study from Finland identified 842 patients aged 35-74 years with bronchiectasis and followed them for 8-13 years. These patients were also compared with asthma and COPD controls. The mortality was not found to be significantly different among the 3 groups (bronchiectasis, asthma, COPD) with mortality rates of 28%, 20%, and 38% respectively. Currently, mortality is more often related to progressive respiratory failure and cor pulmonale than to uncontrolled infection. Life-threatening hemoptysis may also occur but is uncommon (Emmons Bronchiectasis. In: WebMD Hollingsworth, H M (Ed) 2008). Bronchiectasis is the prototypical disease for which secretion loosening or thinning combined with enhanced removal techniques should be salutary, although large population and long-term studies of efficacy are lacking. This is particularly important as tenacious secretions and mucous plugs are frequently present. Potential approaches include hydration, nebulization with saline solutions and mucolytic agents, mechanical techniques, bronchodilators, and anti-inflammatory therapy. (Barker, A. F. Treatment of bronchiectasis. In: UpToDate, King T E (Ed), UpToDate, Wellesley, Mass., 2008.) Treatment of bronchiectasis is aimed at controlling infection and improving bronchial hygiene. Since infection plays a major role in causing and perpetuating bronchiectasis, reducing the microbial load and attendant mediators is a cornerstone of therapy (Barker, A. F. Treatment of bronchiectasis. In: UpToDate, King T E (Ed), UpToDate, Wellesley, Mass., 2008).
Treatment strategies including daily oral antibiotic treatment, daily or three times weekly use of a macrolide antibiotic treatment, aerosolization of an antibiotic, and intermittent intravenous antibiotics have not been established in long-term studies (Barker, A. F,). Several antibiotic treatment strategies are expensive and require extra equipment and personnel and only target part of the pathophysiology of the disease. Other treatments include physiotherapy, hydration with oral liquids and nebulization with hyperosmolar or mucolytic agents, bronchodilators, anti-inflammatory medications such as corticosteroids, and surgery. (Barker, A. F.) Thus, the treatments for bronchiectasis are limited in their ability to affect key pathophysiologies of the disease.
Alpha-1-Antitrypsin Deficiency (AATD)
AATD is a common inherited genetic disorder which severely affects up to 100,000 people in the US alone. (Campos, M A et al. Chest, 128:1179, 2005). An important physiological role for alpha-1-antitrypsin (AAT) is to protect lung elastin from degradation by serine proteases such as neutrophil elastase, which is repeatedly produced by lung tissues as a normal immune response to inhaled airborne pathogens. Low levels of AAT and/or secretion of defective AAT can lead to an imbalance between antiproteases and their target serine proteases, leading to tissue damage by these potent degrading enzymes (Koehlein, T et al. Am J Med., 121:3-9, 2008).
A further aspect of the secretion of defective protein is the loss of the anti-inflammatory properties exerted by the normal protein. AAT is mainly produced in the hepatocytes, with the most common inherited AAT defect giving rise to an accumulation of abnormal protein in these cells, often resulting in cell damage (Lomas, D A, et al. Nature, 357:605, 1992). In the lung, the alveoli show low levels of functional AAT, often leading to an imbalance between antiprotease and protease, and consequential tissue destruction. While the correlation between the severity of the protein deficiency and resultant disease is somewhat variable (Silverman, E K et al. Ann Intern Med, 111:982, 1989), AATD is associated with increased risk for COPD, emphysema, asthma, chronic bronchitis, and brochiectasis in the lung, as well as cirrhosis, hepatitis, hepatocarcinoma or liver failure.
A major risk factor for COPD and emphysema in AATD patients is smoking, thus a smoking cessation program is a critical first-line defense against the progression of disease. Current available therapies for COPD and emphysema include use of long acting beta-agonists and anticholinergics to promote bronchorelaxation, treatment with steroids to reduce inflammation, or supplementation of AAT levels with AAT isolated from the pooled blood of human donors. (Koehlein, T et al. Am J Med., 121:3-9, 2008). A recombinant form of AAT is not yet available for clinical use (Trexler, M M, et al. Biotechnol Prog, 18:501, 2002). However, as none of these therapies are particularly effective, there is an unmet medical need for improved drugs for the treatment of AATD induced lung disease.
Rhinitis
Rhinitis is irritation and inflammation of the mucosal lining of the nose, which may be caused by allergies or other factors such as cigarette smoke, changes in temperatures, and exercise and stress. The resulting irritation and inflammation generate excessive amounts of mucus producing a runny nose, nasal congestion, and post-nasal drip. Rhinitis is a global health concern and is often combined with asthma in determining morbidity due to respiratory diseases. It is a complex disease affecting approximately 20% of the population. Rhinitis occurs in different types: allergic or atopic rhinitis including seasonal and perennial forms. The mechanism of perennial rhinitis with non-allergic triggers is not well understood. It is an allergy-like condition but not triggered by allergens. (Braunstahl et al. Current Opinion in Pulmonary Medicine 2003, 9:46-51). Idiopathic non-allergic rhinitis or vasomotor rhinitis is characterize by nasal congestion and post nasal drip in responses to temperature and humidity changes, smoke, odors, and emotional upsets. In general rhinitis is characterized by a symptoms complex that consists of any combination of the following: sneezing, nasal congestion, nasal itching and irritation, sneezing and watery rhinorrhea, frequently accompanied by nasal congestion. Perennial allergic rhinitis clinical symptoms are similar, except that nasal blockage may be more pronounced. Each type of allergic rhinitis may cause additional symptoms such as itching of the throat and/or eyes, excessive tearing, and edema around the eyes. These symptoms may vary in intensity from the nuisance level to debilitating. Other types of rhinitis present the same symptoms (Kim et al. Current Opinions in Otolaryngology & Head and Neck Surgery 2007, 15: 268-273).
Rho-kinase (RHO KINASE) regulates endothelial permeability by reorganization of the actin-based cytoskeleton and contraction of endothelial cells, resulting in the formation of an intercellular gap. (Walsh et. al. Gastroenterology 2001. 121(3): 566-579). Rho-kinase (RHO KINASE) regulates also regulates epithelial permeability by reorganization of the actin-based cytoskeleton and contraction of epithelial cells (Sawafuji et al. Am J Physiol Lung Cell Mol Physiol 289: L946-L953).
Rhinosinusitis
Rhinosinusitis, an inflammation of the sinus cavity, is the most commonly diagnosed chronic illness in the United States. The name of the disease “rhinosinusitis” is preferred over sinusitis as the inflammation of the sinuses rarely occurs without inflammation of the nasal mucosal at the same time. The disease affects over thirty million people in the United States alone. The treatments for rhinosinusitis are costly, exceeding $200 million per year. This illness is detrimental to both the overall quality of life and economic welfare of sufferers. Currently there is no universally accepted treatment for rhinosinusitis; therefore a need to identify new molecular pathways targeting the disease exists.
Sinusitis is the inflammation of the mucus membranes involving the paranasal sinuses, nasal cavity, and underlying bone. A leading theory suggests that exposure to allergens induces inflammation in the small channels of the ostiomeatal complex (OMC), which results in mucosal edema and ultimately impaired mucociliary clearance of the sinus ostia leading to blockage. As a result the trapped mucus becomes a breeding ground for bacteria and other microorganisms which can lead to infection. Common symptoms include pain varying from forehead to teeth, cheeks, ears, and neck, nasal drainage or postnasal drip and decreased sense of smell (Metson, R. et al. Chronic rhinosinusitis, In: UpToDate, Calderwood, S B (Ed), UpToDate, Wellesley, Mass., 2008).
Depending upon the durations of symptoms, rhinosinusitis may be classified as acute, sub acute, or chronic. Chronic sinusitis has long-term effects that could last over twelve weeks and accounts for >90% of all cases of rhinosinusitis The effects of chronic rhinosinusitis are debilitating even when compared to other chronic illnesses such as heart failure or pulmonary disease because it has potential to cause physical and physiological impairment (Metson, R. et al. Chronic rhinosinusitis, In: UpToDate, Calderwood, S B (Ed), UpToDate, Wellesley, Mass., 2008).
Other Respiratory Diseases Characterized by Airway Inflammation, Lung Tissue Edema, Bronchoconstriction and/or Airway Hyperreactivity
Primary ciliary diskinesia (PCD), pneumonia, and bronchiolitis caused by agents other than RSV are respiratory disorders with medical need unmet by existing treatments and at least one of the following pathophysiologies accompanying these diseases: increased airway inflammation, lung tissue edema and/or bronchoconstriction or airway hyperreactivity. Pneumonia is a cause of significant morbidity and/or mortality in developed and developing world with World Health Organization estimates of 150.7 million cases worldwide every year. There is a variety of etiologic agents with large portion being viral and bacterial (i.e. M. pneumoniae or Influenza A and B). Pneumonia is accompanied by lung inflammation and lung tissue edema. PCD is a rare genetic mutation leading to defect in cilia. The main consequence is decreased ciliary clearance and increased airway inflammation due to recurrent respiratory infections and mucus accumulation in the airway. Bronchiolitis is a common cause of illness and hospitalization in infants and children younger than two years. Bronchiolitis is broadly defined as an illness characterized by wheezing and airways obstruction that is caused by infection with a viral or, less commonly, a bacterial pathogen resulting in inflammation of the small airways/bronchioles. Although respiratory syncytial virus (RSV) is the most common cause, parainfluenza virus, human metapneumovirus, influenza virus, adenovirus, rhinovirus, coronavirus, and human bocavirus are other infectious agents know to cause bronchiolitis.
OB/BOOP Due to Lung Transplantation and HSCT
Obliterative bronchiolitis (OB) is characterised by the onset of new air flow obstruction due to functional obstruction of the bronchioles. OB is a common late noninfectious pulmonary complication following both lung transplantation and allogeneic haematopoietic stem cell transplantation (HSCT) with an incidence of 50-60% in patients who survive for 5 years after lung transplantation and 0-48% following HSCT. OB accounts for more than 30% of all deaths occurring after the third postoperative year for lung transplant patients. The mortality rate in patients with OB following HSCT varies from 14-100%, with a median of 65%. Graft versus host disease is an established risk factor for OB after lung transplantation and HSCT. The histopathologic features of OB suggest that injury and inflammation of epithelial cells and subepithelial structures of small airways lead to excessive fibroproliferation, seemingly due to ineffective epithelial regeneration and aberrant tissue repair. The respiratory symptoms of OB include dry cough, dyspnea, and wheezing. Lung biopsies show small airway involvement with fibrinous obliteration of the lumen. BAL shows neutrophilic and/or lymphocytic inflammation. Despite treatment with corticosteroids and immunosuppression, improvement in lung function is noted in only 8% to 20% of patients with OB. Most patients with OB progress to respiratory failure, and some patients develop bronchiectasis with frequent bacterial exacerbations (Afessa B, Bone Marrow Transplantation 28: 524-434, 2001; Nicod L P, Proc Am Thorac Soc 3: 444-449, 2006; Estenne M, Am J Respir Crit Care Med 166: 440-444, 2002).
Bronchiolitis obliterans organizing pneumonia (BOOP) is a complication of both lung transplantation and HSCT and is defined by the patchy distribution of plugs of granulation tissue that fill the lumens of the distal airways, extending into the alveolar ducts and alveolar sacs in association with chronic interstitial inflammation. Organizing pneumonia results from alveolar epithelial injury with subsequent intra-alveolar fibrosis, angiogenesis and inflammation. Clinically, patients present with fever, cough, dyspnea, and crackles on physical examination with onset between 1 and 13 months following HSCT. The clinical spectrum of BOOP ranges from a mild illness to respiratory failure and death. BOOP usually responds well to corticosteroid treatment, however, frequent relapse occurs and new therapeutic options are needed to treat BOOP. (Cordier et al, Eur Resp J, 28:422-446, 2006; Freudenberger T D et al. Blood, 102:3822-3828, 2003; Travis W D et al. Am J Respir Grit Care Med 165: 277-304, 2002).
The therapeutic options for BO/BOOP include corticosteroids and immunosuppressive agents. However, these treatments are often of limited efficacy and new treatment options are needed to address BO/BOOP following lung transplantation and HSCT.
Non-IPF Idiopathic Interstitial Pneumonia
The idiopathic interstitial pneumonias (IIPs) are a group of interstial lung diseases (ILD, also know as diffuse parenchymal lung disease or DPLD) that result from damage to the lung parenchyma by varying patterns of inflammation and fibrosis. The interstitium includes the space between the epithelial and endothelial basement membranes and it is the primary site of injury in the IIPs. However, these disorders frequently affect not only the interstitium, but also the airspaces, peripheral airways, and vessels along with their respective epithelial and endothelial linings. The IIPs described comprise a number of clinicopathologic entities, which are sufficiently different from one another to be designated as separate disease entities. The idiopathic interstitial pneumonias include the entities of idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP), acute interstitial pneumonia (AIP), respiratory bronchiolitis-associated interstitial lung disease (RB-ILD), desquamative interstitial pneumonia (DIP), and lymphocytic interstitial pneumonia (LIP). Several clinical findings common to the IIPs are exertional dyspnea or cough, bilateral diffuse interstitial infiltrates on chest radiographs, physiological and gas exchange abnormalities including a decreased carbon monoxide diffusion capacity (DLCO) and an abnormal alveolar-arteriolar PO2 difference, and histopathologic abnormalities of the pulmonary parenchyma that are characterized by varying marked inflammation, fibrosis and remodeling (Raghu G et al. Clin Chest Med 25:409-419, 2004; Travis W D et al. Am J Respir Crit Care Med 165: 277-304, 2002). The clinical prognosis of these diseases ranges from mild illness to respiratory failure and death. Therapies for the IIPs include corticosteroids and immunosuppressive agents but current treatments are variably effective and new treatment options are needed to treat these diseases.
ILD Other than IPF, Non-IPF IIP, and OB/BOOP
Interstitial Lung Disease (ILD), also known as diffuse parenchymal lung disease (DPLD), represent a variety of disorders that lead to diffuse remodeling, architectural damage to normal lung tissue and inflammation that lead to progressive loss of lung function. In addition to the inflammation and fibrosis that is often seen in the lung parenchyma in ILD, the airways and the vasculature may also be prominently affected. The ILDs can be classified into 7 main groups: iatrogenic or drug-induced; occupational or environmental; granulomatous diseases including pulmonary sarcoidosis collagen-vascular disease; unique entities such as alveolar proteinosis, Langerhans cell granulomatosis, and lymphangioleiomyomatosis; idiopathic interstitial pneumonias including interstitial pulmonary fibrosis (IPF); and inherited disorders such as tuberous sclerosis, neurofibromatosis, metabolic storage disorders and Hermansky-Pudlak syndrome. The most prominent forms of ILD are IPF and pulmonary sarcoidosis. Several clinical findings are common to the ILDs: exertional dyspnea or cough; bilateral diffuse interstitial infiltrates on chest radiographs; physiological and gas exchange abnormalities including a decreased carbon monoxide diffusion capacity (DLCO) and an abnormal alveolar-arteriolar PO2 difference; and histopathologic abnormalities of the pulmonary parenchyma that are characterized by varying degrees of inflammation, fibrosis and remodeling. The incidence of ILD is estimated to be 31.5 per 100,000/yr in males and 26.1 per 100,000/yr in females and the clinical prognosis of these diseases range from mild illness to respiratory failure and death (Raghu G et al. Clin Chest Med 25:409-419, 2004). The standard therapies for ILD include corticosteroids and immunosuppressive agents but current treatments are variably effective depending on the specific disease entity being treated and new treatment options that suppress inflammation and prevent fibroblast and myofibrobalst proliferation are needed to treat these diseases (Kim et al. Ther Adv Respir Dis 2:319-338, 2008).
There is a need for an effective or improved method for treating ophthalmic diseases such as allergic conjunctivitis, corneal hyposensitivity and kerotopathy, dry eye disease, proliferative vitreal retinopathy, macular edema and degeneration, blepharitis and disorders in which intraocular pressure is elevated, such as primary open-angle glaucoma. There is a need for an effective or improved method for treating pulmonary diseases such as asthma, COPD, respiratory tract illness caused by respiratory syncytial virus infection, PAH, LAM, idiopathic pulmonary fibrosis, ARDS and VILI, CF, bronchiectasis, AATD, rhinitis, rhinosinusitis, PCD, pneumonia, bronchiolitis caused by agents other than RSV, OB/BOOP due to lung transplantation or HSCT, non-IPF IIP and ILD other than IPF, non-IPF IIPs and OB/BOOP.